Methylcrotonyl-CoA carboxylase 2 supports leucine catabolism to promote mitochondrial biogenesis and alleviate cisplatin-induced acute kidney injury

Article information

Korean J Nephrol. 2025;.j.krcp.24.169
Publication date (electronic) : 2025 January 9
doi : https://doi.org/10.23876/j.krcp.24.169
1Department of Urology, The Fifth Affiliated Hospital of Sun Yat-Sen University, Zhuhai, China
2Guangdong Provincial Key Laboratory of Biomedical Imaging, The Fifth Affiliated Hospital of Sun Yat-Sen University, Zhuhai, China
Correspondence: Yingbo Dai Department of Urology, The Fifth Affiliated Hospital of Sun Yat-sen University, No. 52 Mei Hua Dong Road, Zhuhai 519000, China. E-mail: daiyingbo@126.com
*Hu Li and Kangqiang Weng contributed equally to this study work as co-first authors.
Received 2024 June 26; Revised 2024 September 1; Accepted 2024 September 16.

Abstract

Background

Cisplatin is widely used in clinical practice, but its nephrotoxicity severely limits its use. Previous studies have shown that cisplatin-induced acute kidney injury (AKI) is closely related to mitochondrial damage and that alleviating mitochondrial dysfunction can alleviate cisplatin-induced AKI. Methylcrotonyl‑CoA carboxylase 2 (MCCC2) is mainly located in mitochondria, where it catalyzes the catabolism of leucine and maintains mitochondrial function; however, the role of MCCC2 in cisplatin-induced renal injury has not yet been studied.

Methods

In vitro, the expression of MCCC2 was manipulated by transfecting HK-2 cells with lentiviruses, and changes in the acetoacetate content, cell viability, apoptosis, oxidative stress, mitochondrial function, and mitochondrial biogenesis were evaluated. In vivo, MCCC2 overexpression was manipulated by adeno-associated viruses, and serum and kidneys were collected for subsequent experiments to detect changes in renal function, tissue damage, apoptosis, oxidative stress, mitochondrial damage, and mitochondrial biogenesis.

Results

We found that MCCC2 was downregulated in cisplatin-induced AKI models. In vitro, leucine catabolism was inhibited by cisplatin, while overexpression of MCCC2 supported leucine catabolism, upregulated peroxisome proliferator-activated receptor gamma coactivator 1-alpha expression, promoted mitochondrial biogenesis, improved mitochondrial function, and alleviated cisplatin-induced apoptosis and oxidative stress in HK-2 cells. In contrast, the knockdown of MCCC2 exacerbated these effects, while leucine deprivation reversed the effects of MCCC2 overexpression on mitochondrial function and biogenesis. In vivo, the overexpression of MCCC2 promoted mitochondrial biogenesis, maintained the integrity of the mitochondrial structure and function, and alleviated cisplatin-induced AKI.

Conclusion

MCCC2 supported leucine catabolism and promoted mitochondrial biogenesis, providing a new therapeutic strategy for cisplatin-induced AKI.

Introduction

Acute kidney injury (AKI) is a common clinical syndrome that refers to the sudden loss of renal function, as evidenced by an increase in the creatinine level and a decrease in urine output [1]. Previous studies have reported a prevalence rate of AKI of approximately 10% to 15% in hospitalized patients, with even higher rates in some cases [2]. Without intervention, patients eventually develop chronic kidney disease (CKD) or end-stage renal disease. Cisplatin is widely used to treat malignant tumors; however, it is a double-edged sword, offering both therapeutic benefits and serious side effects, such as nephrotoxicity, ototoxicity, and neurotoxicity [35]. Among these, nephrotoxicity is the most serious. AKI occurs in approximately one-third of all patients treated with cisplatin [6], which greatly reduces their quality of life and restricts the clinical application of cisplatin. Although there has been a large number of studies related to cisplatin-induced AKI, there is still a lack of effective therapy for it. Therefore, addressing this issue is of great importance.

After entering the body, cisplatin accumulates in large quantities within renal tubular epithelial cells, which subsequently leads to cell damage and death [7,8]. Studies have shown that cisplatin binds to DNA to form adducts [9], ultimately leading to cell apoptosis and necrosis. Notably, the ability of cisplatin to bind to mitochondrial DNA (mtDNA) is approximately 300 to 500 times greater than that of nuclear DNA [10,11]. The kidneys are rich in mitochondria and have the second-highest mitochondrial content in the human body [12]. Therefore, an increasing number of studies have suggested that mitochondrial dysfunction plays an important role in cisplatin-induced AKI, which is mainly characterized by a reduction in the mitochondrial number and a decrease in mitochondrial function [13].

Mitochondria are the main site for the production of energy and reactive oxygen species (ROS), and their impairment leads to the lack of energy and an increase in ROS production, which results in oxidative stress and exacerbates mitochondrial damage, leading to the development of AKI [14,15]. In addition, studies have shown that apoptosis is the main mode of cell death in cisplatin-induced AKI, and mitochondrial dysfunction is one of the main mediating factors of apoptosis [16]. Therefore, numerous lines of evidence suggest that alleviating mitochondrial dysfunction is important for mitigating cisplatin-induced AKI [17,18]. Numerous studies have suggested that promoting mitochondrial biogenesis can alleviate mitochondrial dysfunction [1921]. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a major regulator of mitochondrial biogenesis, and in cisplatin-induced AKI, PGC-1α is significantly inhibited, whereas its activation alleviates cell damage [19].

Methylcrotonyl‑CoA carboxylase 2 (MCCC2) is located in mitochondria, and it encodes a subunit of 3-methylcrotonyl coenzyme A carboxylase (MCC), which is known to be an enzyme involved in leucine catabolism [22]. The deficiency of MCC leads to an autosomal recessive disease, with affected patients suffering from hypoglycemia, severe acidosis, hypotonia, seizures, and varying degrees of developmental delay [23]. In addition, MCCC2 is associated with the development of a variety of cancers, such as breast, prostate, and hepatocellular carcinomas [2426]. The relationship between MCCC2 and mitochondria affects the mitochondrial membrane potential, mitochondrial fusion, and adenosine triphosphate (ATP) production [25,27,28]. However, the role of MCCC2 has not yet been reported in AKI, and our study showed that in cisplatin-induced AKI, MCCC2 supported leucine catabolism, promoted the expression of PGC-1α, increased mitochondrial biogenesis, alleviated mitochondrial dysfunction, and attenuated renal tubular cell injury.

Methods

Ethics statement

No human specimens were used in this experiment. All animal procedures were approved by the Institutional Animal Care and Utilization Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (Guangdong, China) (No. 00415).

Bioinformatics

The dataset GSE142173 for cisplatin-induced AKI was obtained from Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), and differential analyses were performed using the DESeq2 package in the R software, the selection of final differentially expressed genes (DEGs) was restricted by padj <0.05 and |log2FoldChange| >1. The list of mitochondrial genes was obtained from the MitoCarta 3.0 database (https://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the DEGs of GSE142173 dataset and the mitochondrial genes were performed with the clusterProfiler R package, data were visualized using the ggplot2 package in R software. Pathways of valine, leucine, and isoleucine degradation were extracted separately from the two gene lists, and the intersection of the two was analyzed.

Cell culture and treatment

Human renal tubular epithelial cells (HK-2 cells) obtained from the Procell Life Science & Technology, which were culture in Dulbecco’s modified Eagle medium-F-12 (Gibco, Thermo Fisher Scientific) with 10% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 g/mL streptomycin (Gibco). The cells were cultured in a humidified 5% CO2 incubator maintained at 37 °C. HK-2 cells were identified using short tandem repeat profiling. All cells were tested to confirm they were free of mycoplasma contamination. Cisplatin (P4394) was obtained from Sigma-Aldrich. HK-2 cells were treated with cisplatin for 24 hours to construct a renal tubular cell injury model.

Lentiviruses for this study were designed and synthesized by GeneChem Company in Shanghai, China, and were added to the culture medium when the cell density reached 50%. Lentiviruses for this experiment included: overexpression lentiviruses of MCCC2 (oeMCCC2), knockdown lentiviruses of MCCC2 (shMCCC2), and their empty vector viruses (oeNC and shNC). The shRNA sequences are as listed:

shMCCC2-1: 5′-GGGCCCAAGAAATTGCCATGC -3′

shMCCC2-2: 5′-GGATCTTGGAGGTGCTGATCT -3′

shMCCC2-3: 5′-GCAGATTCACTGAGTTCAAAG -3′

Cell viability assay

The cell counting kit-8 (CCK-8) assay was conducted to detect the viability of HK-2 cells. Briefly, cells were seeded in 96-well plates at 6,000 cells/well; then, the plates were incubated at 37 °C and 5% CO2. After the cells adhered, they were treated with cisplatin for the scheduled time. Then, removing the medium before adding the 200-μL CCK-8 solution (Dojindo Laboratories) and incubating at 37 °C for 2 hours, the absorbance value of each well was measured at 450 nm using a microplate reader.

Quantitative real-time polymerase chain reaction

The Total RNA Kit (Omega Bio-tek) was used to extract total RNAs from HK-2 cells following the manufacturer’s protocol. RNAiso Plus (Takara) was used to extract kidney messenger RNA (mRNA). Complementary DNA synthesis was performed using the HiScript III RT SuperMix for the quantitative polymerase chain reaction (qPCR) kit (Vazyme) according to the manufacturer’s instructions. Real-time qPCR (RT-qPCR) was performed using SYBR Green mix (Vazyme) in Applied Biosystems 7500 RT-PCR system. The gene primers used are listed in Supplementary Tables 1 and 2 (available online). The mRNA expression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase and calculated using the 2–ΔΔCt method.

Flow cytometry

The apoptosis of HK-2 cells was evaluated by flow cytometry. Cells were seeded in six-well plates and were incubated at 37 °C and 5% CO2 at constant temperature. After treatment, digest with trypsin and centrifuge to obtain cell precipitate. Cells were washed with phosphate-buffered saline (PBS) and then resuspended in binding buffer. Annexin V/PI (Vazyme) was used to stain the cells. The stained cells were analyzed by flow cytometry (Beckman Coulter).

Western blot

The kidney tissues were removed and weighed, and the cells were washed with PBS three times and then lysed with radio-immunoprecipitation assay buffer (Cwbio) containing protease inhibitors (Cwbio) for 30 minutes on ice. Mitochondrial protein was extracted using a mitochondrial protein extraction kit (Beyotime Biotechnology). The protein concentration of the sample was measured by a bicinchoninic acid protein assay kit (Biosharp) and denatured by incubation at 100 °C for 10 minutes. An equivalent quantity of protein was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Bio-Rad) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). PVDF membranes were placed in 5% skim milk diluted with Tris-buffered saline with Tween 20 buffer and incubated for 1 hour and then placed in primary antibody and incubated overnight at 4 °C. After incubation, horseradish peroxidase (HRP)-conjugated secondary antibody was used at room temperature for 1 hour. Proteins were visualized using enhanced chemiluminescence (Millipore) and quantified using ImageJ software. The antibodies used for this experiment were as follows: MCCC2 (Proteintech; 12117-1-AP, 1:1,000), cytochrome c oxidase IV (COX IV) (Proteintech; 66110-1-lg, 1:10,000), cleaved caspase-3 (Cell Signaling Technology; 9664S, 1:1,000), PGC-1α (Proteintech; 66369-1-lg, 1:10,000), β-actin (Abclonal; AC026, 1:20,000), secondary antibody anti-Mouse (Abclonal; AS003, 1:10,000), secondary antibody anti-Rabbit (Abclonal; AS014, 1:10,000).

Intracellular ROS levels determination

The level of ROS in HK-2 cells was detected by ROS Detection Reagents (Invitrogen). Briefly, cells were stained by CM-H2DCFDA according to the manufacturer’s instructions and then detected by an integrated fluorescence microscope imaging system (Keyence) and ImageJ software was used to evaluate the fluorescence intensity.

Measurement of mitochondrial membrane potential

The measurement of mitochondrial membrane potential was detected by JC-1 staining (Beyotime Biotechnology) following the manufacturer’s instructions. HK-2 cells were stained with JC-1 staining solution and then incubated at 37 ℃ with 5% CO2 for 20 minutes, washed with PBS. Images were visualized and captured under the integrated fluorescence microscope imaging system (Keyence). The ratio of red to green fluorescence intensity was calculated using ImageJ software.

Measurement of mitochondrial ROS

The level of mitochondrial ROS (mitoROS) was detected by MitoSOX (Dojindo Laboratories). Briefly, cells or frozen section were incubated in MitoSOX at 37 ℃ for 30 minutes, images were visualized and captured under an integrated fluorescence microscope imaging system (Keyence) and ImageJ software was used to evaluate MitoSOX intensity.

Measurement of adenosine triphosphate

The renal tissue and cells ATP levels were determined by chemiluminescence assay using an ATP assay kit (Beyotime Biotechnology) according to the manufacturer’s instructions. We collected tissue or cell lysates with lysis buffer, centrifuged at 12,000×g for 5 minutes at 4 °C, and then transferred the supernatant and luciferase reagents to a 96-well plate. Luminescence was detected using a multifunctional microplate reader (PerkinElmer). The ATP levels were normalized by protein concentration.

Mitochondrial DNA quantification

Total DNA was harvested from treated cells or mice kidneys using FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme). The mtDNA copy number was expressed as the mtDNA/nuclear DNA ratio. The mtDNA levels were determined by comparing the cycle threshold of mitochondrial genes (ND1) and nuclear genes (β-actin). Relative expression of mtDNA was calculated using the 2−ΔΔCt method. The sequences of the primers are given in Supplementary Tables 1 and 2 (available online).

Immunofluorescence

HK-2 cells were fixed and permeabilized; and the tissue slides were dewaxed, rehydrated, and subjected to antigen retrieval, followed by blocking for 1 hour. The sections were then incubated with primary antibody dilution overnight at 4 °C. After three times washing, secondary antibody dilutions were used to incubate the sections for 1 hour at room temperature. To see the nuclei, cells were then labeled with 4′,6-diamidino-2-phenylindole (DAPI) for 10 minutes at room temperature. Finally, the sections were analyzed by an integrated fluorescence microscope imaging system (Keyence) and fluorescence intensity analysis was performed by ImageJ software. For MitoTracker (Beyotime Biotechnology) staining, after HK-2 cells were cultured to the appropriate density, the original medium was replaced with a pre-warmed medium containing the MitoTracker probe and incubated for 30 minutes at 37 °C in an incubator. After staining, the cells were gently washed three times with PBS to remove unbound dye. Subsequently, cells were fixed with 4% paraformaldehyde for 15 minutes and washed again with PBS. The distribution and morphology of mitochondria in the stained cells were observed under a fluorescence microscope. The antibodies used were as following:MCCC2 (Abclonal; A15181, 1:100), COX IV (Proteintech; 66110-1-lg, 1:100), translocase of outer mitochondrial membrane 20 (TOMM20) (Proteintech; 11802-1-AP, 1:250), 4-hydroxynonenal (4-HNE) (Abclonal; A24456, 1:100), 594-conjugated Goat anti-Mouse (Proteintech; SA00013-3, 1:250), 594-conjugated Goat anti-Rabbit (Abclonal; AS039, 1:100), 488-conjugated Goat anti-Rabbit (Abclonal; AS053, 1:250).

Immunohistochemistry

Immunohistochemistry was performed following the instructions of the SABC anti-rabbit-POD kit (SA1028; Boster Bioengineering). Briefly, paraffin sections were dewaxed and rehydrated, and antigenically repaired in citrate buffer. Sections were treated with 3% H2O2 to block endogenous peroxidase activity and subsequently blocked with 5% goat serum. Afterward, the sections were incubated with specific primary antibodies at 4 °C overnight. After being washed with PBS, the sections were incubated with HRP-labeled secondary antibody at room temperature for 1 hour. DAB (3,3'-diaminobenzidine) was used for chromatography, and then, the sections were restained with hematoxylin, and finally blocked with neutral gum after dehydration and hyaluronidation. The staining results were observed under a light microscope and images were captured and analyzed. The antibodies used were as following: MCCC2 (Abclonal; A15181, 1:100), secondary antibody anti-Rabbit (Abclonal; AS014, 1:100).

Animal experiments

All male mice (C57BL/6J; 8–10 weeks, 20–25 g) were purchased from the Guangdong Provincial Medical Laboratory Animal Center. The mice were provided with free access to food and water. They were allowed to acclimate to their new environment for a week before starting the study. Randomization and blinding for animal studies followed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for animal studies. The mice were divided into multiple groups (each group had six mice).

For the induction of AKI, cisplatin was dissolved in PBS to a final concentration of 1 mg/mL, and cisplatin solution was intraperitoneally injected into the mice at the dose of 25 mg/kg. In contrast, the control group was injected with the same volume of PBS based on body weight. Mice were anesthetized by inhalation of isoflurane and sacrificed 72 hours after cisplatin injection, and then harvested the kidneys and blood samples.

Mouse MCCC2 overexpression adeno-associated virus (AAV, the type of AAV is AAV-DJ) was obtained from GeneChem Company. To manipulate the expression of MCCC2 in mice. The mice were anesthetized and maintained by isoflurane. The mice were warmed with heating pads to maintain body temperature. A small incision was made on each side of the middle of the back of the mice to expose the kidneys under aseptic conditions, and the kidneys were gently lifted to the outside of the incision, fully exposing the whole kidneys to the outside of the body. According to the manufacturer’s instruction, the kidney cortex was carefully pierced with a microsyringe (100 μL, Bolige) and 10 μL of AAV with a concentration of 1 × 1012 vg/mL was slowly injected from the upper, lower, anterior, and posterior sites for each kidney, which means that each kidney was injected four times (AAV 40 μL), for a total of eight injections for each mouse (AAV 80 μL). After the injection was completed, the needle was briefly left in place and then withdrawn, cotton swabs were compressed to stop bleeding, and the kidney was carefully returned to its place after checking that there was no obvious bleeding in the kidney. The incorporated AAV in this study were: MCCC2 overexpression AAV (AAV-MCCC2) and empty vector AAV (AAV-NC).

Measurement of creatinine and urea nitrogen in blood serum

The blood samples of mice were collected and placed for 40 minutes at room temperature (25 °C) and then centrifuged at 4,000 rpm (1,500×g) for 20 minutes at 4 °C to obtain the serum sample. The level of creatinine in serum was detected using the Creatinine Assay Kit (Bioassay) and urea nitrogen was using the Urea Assay Kit (Applygen), according to the manufacturer’s instructions.

Histological analysis

The kidney tissues were fixed in 4% paraformaldehyde overnight and embedded in paraffin wax. Then, sectioned and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). The tissue morphology was observed and imaged with an optical microscope. Histopathology was assessed by grading tubular necrosis, loss of brush border, cast formation, and tubular dilatation. A minimum of five fields (400×) for each kidney tissue were examined and scored for tubular injury. The structural changes and damage characteristics of the renal tubules were observed, and the results were scored and recorded according to the percentage of damaged tubules to the total renal tubules in the field of view. A score of 0 to 4 was given for tissue pathological damage: 0, no abnormalities; 1+, <25%; 2+, 25% to 50%; 3+, 50% to 75%; 4+, >75%. The mean score for each field of view was calculated and statistically analyzed to assess differences between the different groups.

Measurement of malondialdehyde levels

The kidney tissues were removed and weighed, and then homogenized by tissue homogenizer. After centrifugation at 10,000×g for 4 minutes, the supernatant was analyzed using the malondialdehyde (MDA) assay kit (Beyotime Biotechnology) following the manufacturer’s protocol. MDA levels were normalized by protein concentration.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining

The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed using the TUNEL BrightGreen Apoptosis Detection Kit (Vazyme) to identify and quantify apoptosis in renal tissue. Briefly, kidney sections were deparaffinized and rehydrated. Subsequently, the sections were incubated with the TUNEL reagent mixture for 1 hour at 37 °C and washed with PBS three times. Nuclei were stained with DAPI for 10 minutes. Finally, images were observed using fluorescence microscopy. Representative TUNEL staining images showed DAPI in blue and TUNEL staining in green. TUNEL-positive cells were expressed as a percentage of total cells.

Transmission electron microscope

Mouse kidney samples were fixed with 2.5% glutaraldehyde immediately after being harvested and then the cells were fixed with 1% osmic acid solution for 2 hours, dehydrated, and embedded in Epon 812 (SPI). Then samples were sliced into ultrathin sections and stained with uranyl acetate for 25 minutes, followed by lead citrate for 7 minutes. Digital images were obtained with a transmission electron microscope (TEM) (HT-7800, Hitachi).

Measurements of acetoacetate

Acetoacetate levels were measured using the acetoacetate assay kit (Solarbio) following the manufacturer’s instructions. In brief, cell lysates were collected and incubated with the working solution at 37 °C for 10 minutes, and then color developing solution was added and incubated at 37 °C for 20 minutes. The alteration in absorbance (optical density at 450 nm) was measured.

Statistical analysis

Statistical analysis was performed using GraphPad Prism (version 8.0) and quantitative data were presented as the mean ± standard deviation. All assays were conducted at least in triplicate. Statistical differences were determined using the analysis of variance or Student t tests. A p-value of <0.05 was considered to be significant.

Results

MCCC2 was downregulated in cisplatin-induced acute kidney injury

Bioinformatics analysis suggested that the degradation of valine, leucine, and isoleucine (together referred to as branched-chain amino acids, BCAAs) was important in cisplatin-induced AKI (Fig. 1A), and impaired BCAAs catabolism affects mitochondrial function [29], as implied from the fact that a large majority of genes involved in the catabolism of BCAAs are located in mitochondria. The relationship between mitochondrial function and cisplatin-induced AKI has been abundantly demonstrated [30]. Therefore, we selected mitochondrial genes from the MitoCarta 3.0 database for KEGG enrichment analysis (Fig. 1B) and used the intersection of genes involved in BCAAs catabolism with genes of the same function among the DEGs in the GSE142173 dataset from the GEO database (Fig. 1C). The most significantly downregulated gene, MCCC2, was selected as the target for subsequent research (Fig. 1D). We next validated the expression of MCCC2 in cisplatin-induced AKI in vivo and in vitro. In vitro, the results of CCK-8 assay showed that after treating HK-2 cells with different concentrations of cisplatin for 24 hours, the cell viability decreased with the increasing cisplatin concentrations, and its IC50 was 44.61 μM (Supplementary Fig. 1A and B, available online). Based on these data, the in vitro concentration of cisplatin used in subsequent experiments was 40 μM. Interestingly, western blot analysis revealed a dose-dependent decrease in the expression of MCCC2 after treatment with cisplatin (Fig. 1E, F). These findings preliminarily confirmed that MCCC2 expression was downregulated in cisplatin-treated renal tubular epithelial cells. We further validated MCCC2 expression in vivo, and the results of immunofluorescence and immunohistochemistry showed that MCCC2 was highly expressed in renal tubules and was significantly lower in the cisplatin group compared to the control group (Fig. 1GH; Supplementary Fig. 1C and D, available online). Furthermore, western blot analyses showed that the expression of MCCC2 was significantly decreased in the kidneys of mice injected with cisplatin (Fig. 1IJ). To determine the intracellular location of MCCC2, we costained MCCC2 with MitoTracker and found that MCCC2 was abundantly expressed in mitochondria (Fig. 1K). In addition, we isolated mitochondria in HK-2 cells, and the western blot results also showed that MCCC2 was localized in mitochondria (Supplementary Fig. 1E, available online). For subsequent validation, we transduced HK-2 cells with a lentivirus to construct stable cell lines with MCCC2 knockdown and overexpression and confirmed the overexpression and knockdown status of MCCC2 using RT-qPCR and western blotting (Fig. 1LQ).

Figure 1.

MCCC2 was downregulated in cisplatin-induced acute kidney injury.

In vitro, HK-2 cells were treated with different concentrations (0, 10, 20, and 40 μM) of cisplatin. In vivo, mice were injected intraperitoneally with cisplatin (25 mg/kg). (A) Results of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes (DEGs) in the GSE142173 dataset. (B) Results of KEGG enrichment analysis of mitochondrial genes in the MitoCarta 3.0 database (https://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways). (C) Intersection of genes involved in branched-chain amino acids degradation between the GSE142173 DEGs and mitochondrial genes in the MitoCarta 3.0 database. (D) Volcano plot of genes in the intersection group. (E, F) Results of western blot analysis of MCCC2 expression in HK-2 cells (n = 3). (G, H) Results of immunofluorescence staining of MCCC2 in kidney tissue sections from mice (n = 6). Scale bars, 100 μm. (I, J) Results of western blot analysis of MCCC2 expression in kidney tissues of mice (n = 6). (K) To determine the intracellular location of MCCC2, immunofluorescence colocalization of MitoTracker (Beyotime Biotechnology) and MCCC2 was performed. Scale bars, 10 μm. (L, O) Results of real-time quantitative polymerase chain reaction analysis of the efficiency of MCCC2 overexpression and knockdown in HK-2 cells (n = 3). (M, N, P, Q) Results of western blot analysis of the efficiency of MCCC2 overexpression and knockdown in HK-2 cells (n = 3). All the data are presented as the mean ± standard deviation. **p < 0.01, ***p < 0.001.

cis, cisplatin; DAPI, 4′,6-diamidino-2-phenylindole; MCCC2, methylcrotonyl‑CoA carboxylase 2; NS, not significant.

MCCC2 alleviated cisplatin-induced acute kidney injury in vitro

To verify the role of MCCC2 in cisplatin-treated HK-2 cells, we first used a CCK-8 assay to assess cell viability. Notably, cisplatin induced significant HK-2 cell death, and MCCC2 overexpression partially alleviated this effect (Fig. 2A), while MCCC2 knockdown exacerbated it (Fig. 2B). Apoptosis is the main mode of cell death caused by cisplatin-induced renal injury. Western blot analysis revealed that cisplatin significantly increased the level of cleaved caspase-3 in HK-2 cells, and this effect was alleviated or exacerbated depending on the expression status of MCCC2 (Fig. 2CF). In addition, flow cytometry analysis revealed that cisplatin induced significant cell apoptosis, which was alleviated by overexpression of MCCC2 and exacerbated by MCCC2 knockdown (Fig. 2GJ). Many studies have also shown that cisplatin causes intracellular oxidative stress, and therefore, we assessed ROS levels in this study. The data indicated that cisplatin treatment led to an increase in ROS production in HK-2 cells, and MCCC2 overexpression alleviated this effect, while its knockdown exacerbated it (Fig. 2KN). The above data suggested that MCCC2 protected cells against cisplatin-induced AKI in vitro.

Figure 2.

MCCC2 alleviated cell damage in cisplatin-treated HK-2 cells.

HK-2 cells were treated with 40-μM cisplatin for 24 hours. (A, B) Results of the CCK-8 cell viability assay of MCCC2-overexpressing and MCCC2-knockdown HK-2 cells (n = 5). (C–F) Results of western blot analysis of cleaved caspase-3 levels in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells (n = 3). (G–J) Results of flow cytometry analysis of apoptosis levels in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells (n = 3). (K–N) Results of fluorescence detection of ROS levels in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells by using CM-H2DCFDA (n = 3). Scale bars, 100 μm. The data are presented as the mean ± standard deviation. **p < 0.01, ***p < 0.001.

cis, cisplatin; FITC, fluorescein isothiocyanate; MCCC2, methylcrotonyl‑CoA carboxylase 2; PI, propidium iodide; sh, knockdown; oe, overexpression; NC, control.

MCCC2 alleviated cisplatin-induced mitochondrial dysfunction in HK-2 cells

Mitochondrial dysfunction is increasingly recognized as a critical factor in cisplatin-induced renal injury. Therefore, we first examined the changes in the mitochondrial membrane potential in cisplatin-treated HK-2 cells. The results showed that cisplatin caused a significant decrease in the mitochondrial membrane potential, and this decrease was alleviated or exacerbated depending on the expression status of MCCC2 (Fig. 3AD). Mitochondria are energy-producing organelles, and many biological activities depend on normal mitochondrial function. Therefore, we examined the ATP production after cells were treated with cisplatin, and as expected, the data showed that cisplatin reduced ATP production, and this reduction was attenuated by the overexpression of MCCC2 and exacerbated by the knockdown of MCCC2 (Fig. 3E, F). We also assessed the level of mitoROS with MitoSOX and found that cisplatin increased mitoROS production in cells, but this effect was alleviated or exacerbated depending on the expression status of MCCC2 (Fig. 3GJ). Thus, MCCC2 was shown to alleviate mitochondrial dysfunction in cisplatin-induced AKI.

Figure 3.

MCCC2 alleviated mitochondrial dysfunction in cisplatin-treated HK-2 cells.

(A–D) Representative images of mitochondrial membrane potential changes in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells, as determined by using a JC-1 kit and quantitative analysis. Scale bars, 50 μm. (E, F) Adenosine triphosphate (ATP) levels in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells. (G–J) Representative images and the results of quantitative analysis of MitoSOX (Dojindo Laboratories) staining of MCCC2-overexpressing and MCCC2-knockdown HK-2 cells. Scale bars, 50 μm. All the data are presented as the mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

cis, cisplatin; JC-1, J-aggregate-1; MCCC2, methylcrotonyl‑CoA carboxylase 2; sh, knockdown; oe, overexpression; NC, control.

MCCC2 promoted mitochondrial biogenesis in cisplatin-treated HK-2 cells

We then explored the possible mechanism by which MCCC2 alleviated mitochondrial dysfunction in cisplatin-treated HK-2 cells. Western blot analysis revealed that cisplatin caused a decrease in the expression of PGC-1α, and this decrease was alleviated or exacerbated depending on the expression status of MCCC2 (Fig. 4AD). Then, we assessed the transcription levels of several mitochondrial genes. RT-qPCR showed that cisplatin decreased the expression of these genes, but these decreases were ameliorated by the overexpression of MCCC2 and exacerbated by the knockdown of MCCC2 (Fig. 4EL). We next used immunofluorescence to examine the expression of the mitochondrial marker TOMM20, which transports mitochondrial precursor proteins into mitochondria and represents healthy mitochondria. In line with previous findings, cisplatin decreased the expression of TOMM20, and this decrease was modulated by the expression of MCCC2 (Fig. 4MP). mtDNA is also an important indicator of mitochondrial biogenesis. In this study, we demonstrated that cisplatin decreased the number of copies of mtDNA, and this decrease was also alleviated by overexpression of MCCC2 and exacerbated by the knockdown of MCCC2 (Fig. 4Q, R). In conclusion, the above results suggested that MCCC2 alleviated the cisplatin-induced suppression of mitochondrial biogenesis.

Figure 4.

MCCC2 promoted mitochondrial biogenesis in cisplatin-treated HK-2 cells.

(A–D) Results of western blot analysis of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells. (E–L) Results of real-time quantitative polymerase chain reaction analysis of the messenger RNA (mRNA) levels of ATP5a1, NDUFS1, NDUFB8, and SDHB in MCCC2-overexpressing (E–H) and MCCC2-knockdown (I–L) HK-2 cells. (M–P) Representative immunofluorescence images and the results of quantitative analysis of translocase of outer mitochondrial membrane 20 (TOMM20) expression in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells. Scale bars, 50 μm. (Q, R) Mitochondrial DNA (mtDNA) copy numbers in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells. All the data are presented as the mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

cis, cisplatin; MCCC2, methylcrotonyl‑CoA carboxylase 2; sh, knockdown; oe, overexpression; NC, control.

Leucine deprivation reversed the effects of MCCC2 overexpression on mitochondrial function and biogenesis in cisplatin-treated HK-2 cells

The function of MCCC2 is the catabolism of leucine, and we first evaluated changes in the content of acetoacetate, a metabolite of leucine. Cisplatin decreased the level of acetoacetate in cells, while overexpression of MCCC2 increased it (Fig. 5A). Next, to assess whether leucine accumulation exacerbates cisplatin-induced cell damage, we treated HK-2 cells with different concentrations of leucine, and the results showed that even high concentrations of leucine had no effect on cell survival (Fig. 5B), suggesting that the reduction in the levels of leucine metabolites rather than leucine accumulation might exacerbate cell injury. Then, we cultured HK-2 cells in leucine-deprived medium, and the cell viability assay showed that leucine deprivation significantly reversed the protective effect of MCCC2 overexpression against cell death (Fig. 5C). In addition, the ATP content exhibited the same trend (Fig. 5D). We also evaluated the effects of leucine deprivation on mitochondrial biogenesis and showed that the effects of MCCC2 overexpression on PGC-1α expression (Fig. 5E, F), mtDNA content (Fig. 5G), and TOMM20 expression (Fig. 5H, I) were eliminated by leucine deprivation. These results suggested that in cisplatin-induced AKI, the promotion of mitochondrial biogenesis by the overexpression of MCCC2 was dependent on leucine catabolism.

Figure 5.

Leucine deprivation reversed the effects of MCCC2 overexpression on mitochondrial function and biogenesis.

(A) Results of the measurement of the intracellular acetoacetate content (n = 3). (B) HK-2 cells were treated with different concentrations (0, 0.25, 0.5, 1, 2, 3, 4, 6, and 8 μM) of leucine for 24 hours, after which cell viability was assayed (n = 5). (C) Cell viability after leucine deprivation (n = 5). (D) Intracellular adenosine triphosphate (ATP) content after leucine deprivation (n = 3). (E, F) Results of western blot analysis of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression after leucine deprivation (n = 3). (G) Mitochondrial DNA (mtDNA) copy numbers after leucine deprivation (n = 3). (H, I) Results of immunofluorescence staining of translocase of outer mitochondrial membrane 20 (TOMM20) after leucine deprivation (n = 3). Scale bars, 50 μm. All the data are presented as the mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.

cis, cisplatin; DAPI, 4′,6-diamidino-2-phenylindole; leu, leucine; MCCC2, methylcrotonyl‑CoA carboxylase 2; NS, not significant; oe, overexpression; NC, control.

Overexpression of MCCC2 alleviated renal dysfunction and tissue injury in a mouse model of cisplatin-induced acute kidney injury

Next, we validated the function of MCCC2 in vivo. An AAV was used to manipulate the expression of MCCC2 by injecting the virus into the kidneys at multiple sites, 7 days later, the mice were injected intraperitoneally with 25 mg/kg cisplatin, and 72 hours later, the mice were euthanized (Fig. 6A). We examined whether the AAV intervention in the kidneys was successful by western blotting (Fig. 6B, C). Immunofluorescence colocalization of MCCC2 with COX IV further confirmed successful MCCC2 overexpression in kidney mitochondria (Supplementary Fig. 2, available online). The serum and kidney tissues were collected for subsequent renal function tests and morphological evaluation. We found that cisplatin increased the serum creatinine and urea nitrogen levels, whereas the overexpression of MCCC2 alleviated the destruction of renal function (Fig. 6D, E). RT-qPCR of the kidney injury markers KIM-1 and neutrophil gelatinase-associated lipocalin (NGAL) showed that the overexpression of MCCC2 attenuated the increase in the expression of these two markers in the cisplatin groups (Fig. 6F, G). Morphological examination was performed with H&E and PAS staining, and the results showed that the mice treated with cisplatin had obvious dilatation of renal tubules, cell necrosis, tubular formation, and disappearance of the brush border, in contrast, these histological lesions significantly improved after overexpression of MCCC2 (Fig. 6HJ). All these results indicated that the overexpression of MCCC2 attenuated cisplatin-induced AKI in mice.

Figure 6.

Overexpression of MCCC2 alleviated renal dysfunction and tissue injury in mice with cisplatin-induced acute kidney injury (AKI).

(A) Schematic diagram of the establishment of the cisplatin-induced AKI mouse model. (B, C) Results of western blot analysis of the efficiency of MCCC2 overexpression in mouse kidneys. Renal function was assessed by the levels of (D) serum creatinine and (E) urea nitrogen. (F, G) Results of real-time quantitative polymerase chain reaction analysis of the messenger RNA (mRNA) levels of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). (H, I) Representative images of hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining to assess kidney tissue damage (Scale bars, 20 μm). (J) Quantitative data of tubular injury. The data are presented as the mean ± standard deviation (n = 6). **p < 0.01, ***p < 0.001.

AAV, adeno-associated virus; cis, cisplatin; ip, intraperitoneal injection; MCCC2, methylcrotonyl‑CoA carboxylase 2; NC, control.

Overexpression of MCCC2 alleviated cisplatin-induced cell apoptosis and oxidative stress in vivo

To assess the effect of MCCC2 on cisplatin-induced apoptosis in vivo, TUNEL staining was performed. We found that the number of positive cells was much greater in the mice injected with cisplatin, and overexpression of MCCC2 significantly attenuated this effect (Fig. 7A, B). Furthermore, the level of cleaved caspase-3 in renal tissue was assessed, and the data showed that the cisplatin-induced increase in the cleaved caspase-3 level was reversed by the overexpression of MCCC2 (Fig. 7C, D). In addition, cisplatin caused an increase in the MDA level, which is an indicator of oxidative stress, whereas this increase was diminished after overexpression of MCCC2 (Fig. 7E). We also assessed the occurrence of oxidative stress by measuring 4-HNE levels and showed that cisplatin increased 4-HNE production, while overexpression of MCCC2 suppressed this effect (Fig. 7F, G). Overall, the overexpression of MCCC2 ameliorated apoptosis and oxidative stress in mice injected with cisplatin.

Figure 7.

Overexpression of MCCC2 alleviated cell apoptosis and oxidative stress in mice with cisplatin-induced acute kidney injury.

(A, B) Results of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining to detect apoptosis in renal tissues (green represents positive cells). Scale bars, 100 μm. (C, D) Results of western blot analysis of cleaved caspase-3 levels in renal tissues. (E) Malondialdehyde (MDA) levels in renal tissues. (F, G) Results of immunofluorescence staining for 4-HNE in kidney sections. Scale bars, 100 μm. All the data are presented as the mean ± standard deviation (n = 6). ***p < 0.001.

AAV, adeno-associated virus; cis, cisplatin; DAPI, 4′,6-diamidino-2-phenylindole; HPF, high-power field; MCCC2, methylcrotonyl‑CoA carboxylase 2; MDA, malondialdehyde; 4-HNE, 4-hydroxynonenal; NC, control.

Effects of MCCC2 overexpression on mitochondrial function and biogenesis in a mouse model of cisplatin-induced acute kidney injury

We next evaluated the effects of MCCC2 overexpression on mitochondrial structure and function in mice with cisplatin-induced AKI. TEM revealed that the morphology of the proximal renal tubules was significantly altered in the mice treated with cisplatin, as evidenced by the disappearance of cristae, rupture of the membrane, and release of the matrix to the cytoplasm; however, these changes were alleviated by overexpression of MCCC2 (Fig. 8A). In addition, MitoSOX was used to assess the level of mitoROS in renal tissues, and the results showed that overexpression of MCCC2 alleviated the increase in mitoROS production in cisplatin-injected mice (Fig. 8B, C). We also measured the ATP content in mouse kidneys and found that it significantly decreased after injection of cisplatin; however, this decrease was reversed by overexpression of MCCC2 (Fig. 8D). We also evaluated the effects of MCCC2 overexpression on mitochondrial biogenesis in vivo. Western blot analysis suggested that the PGC-1α expression was downregulated in the cisplatin group, but MCCC2 overexpression alleviated this effect (Fig. 8E, F). Then, we validated the expression of mitochondrial genes, and RT-qPCR suggested that overexpression of MCCC2 reversed the suppression of these genes by cisplatin (Fig. 8GJ). In kidney tissues, we also performed TOMM20 staining and showed that the overexpression of MCCC2 alleviated the decrease in TOMM20 expression in the groups treated with cisplatin (Fig. 8K, L). The same effect was observed for the mtDNA copy number (Fig. 8M). The above results suggested that overexpression of MCCC2 protected mitochondrial function and structure from the damage caused by cisplatin in mouse kidneys and promoted mitochondrial biogenesis in the mouse model of cisplatin-induced AKI.

Figure 8.

Effects of MCCC2 overexpression on mitochondrial function and biogenesis in mice with cisplatin-induced acute kidney injury.

(A) Representative transmission electron microscope (TEM) images of the mitochondrial structure in renal tubular epithelial cells. Scale bars, 1 μm. (B, C) Results of MitoSOX (Dojindo Laboratories) staining of kidney sections (n = 6). Scale bars, 100 μm. (D) Adenosine triphosphate (ATP) levels measured in renal tissues (n = 6). (E, F) Results of western blot analysis of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression in kidney tissues (n = 6). (G–J) Real-time quantitative polymerase chain reaction analysis was performed to detect the messenger RNA (mRNA) levels of ATP5a1, NDUFS1, NDUFB8, and SDHB in renal tissues (n = 6). (K, L) Results of translocase of outer mitochondrial membrane 20 (TOMM20) staining in kidney sections (n = 6). Scale bars, 100 μm. (M) Mitochondrial DNA (mtDNA) copy numbers in kidney tissues (n = 6). All the data are presented as the mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.

AAV, adeno-associated virus; cis, cisplatin; DAPI, 4′,6-diamidino-2-phenylindole; MCCC2, methylcrotonyl‑CoA carboxylase 2; NC, control.

Discussion

Cisplatin has a wide range of clinical applications, but its side effects, especially nephrotoxicity, limit its use. Currently, the exact mechanisms of cisplatin-induced AKI are not clear, and exploration of these mechanisms may be important for mitigating the development of AKI. Our research revealed that MCCC2 promoted mitochondrial biogenesis and ameliorated mitochondrial dysfunction by supporting leucine catabolism and alleviating cisplatin-induced AKI.

Bioinformatics analysis revealed that MCCC2 might be closely related to cisplatin-induced AKI. Our results showed that MCCC2 was localized primarily to renal tubules, which are the main site of AKI, and was significantly downregulated in cisplatin-induced AKI models in vivo and in vitro. Studies have shown that MCCC2 is associated with the progression of a variety of cancers. In hepatocellular carcinoma, MCCC2 can promote tumor progression by regulating leucine metabolism [26], and its overexpression correlates with the prognosis of colorectal and breast cancers [24,31]. He et al. [25] demonstrated that MCCC2 promoted the proliferation, migration, and invasion of prostate cancer cells and inhibited apoptosis by regulating the glutamate dehydrogenase 1/p38 mitogen-activated protein kinase signaling pathway. Renal tubular cells are very sensitive to AKI, and many factors can induce cell apoptosis, which is mainly mediated by caspases. Briefly, an extracellular or intracellular stimulus causes cleavage of an initiator caspase, which then cleaves an executioner caspase, leading to DNA fragmentation and cell death. In cisplatin-induced AKI, caspase-3 plays the most important role, and the activation of caspase-3 involves a variety of pathways, such as the mitochondrial pathway, death receptor pathway, and endoplasmic reticulum stress pathway [32]. In our study, we confirmed that cisplatin significantly increased cell apoptosis and cleaved caspase-3 levels, as expected, MCCC2 inhibited both. These findings suggested that MCCC2 could alleviate cisplatin-induced AKI by inhibiting cell apoptosis.

ROS are important contributors to cell damage. Low intracellular levels of ROS are beneficial, whereas high levels of ROS contribute to oxidative stress and cell death [33,34]. The role of oxidative stress in cisplatin nephrotoxicity has been well documented in recent decades, and many studies have shown that some natural antioxidants, such as vitamin C and vitamin E, and the activation of vitamin D receptors can reduce ROS formation to alleviate cisplatin-induced AKI [3537]. It was also reported that MCCC2 modulated the production of ROS in the cell [28]. Therefore, we investigated the effect of MCCC2 on the cisplatin-induced increase in ROS levels by manipulating the expression of MCCC2 in cells and mice, and the results showed that MCCC2 inhibited intracellular oxidative stress.

The kidney is a highly energy-consuming organ that is rich in mitochondria, with proximal tubular epithelial cells having the highest energy demand and being the main site of AKI. In cisplatin-induced AKI, mitochondrial dysfunction plays an important role, and ameliorating mitochondrial dysfunction can alleviate AKI [3840]. As a result, an increasing number of drugs that target mitochondria have emerged [41]. It has been shown that the inhibition of mitochondrial dysfunction suppresses cisplatin-induced apoptosis [42]. The mitochondrial membrane potential is an indicator of mitochondrial function. MCCC2 is a mitochondrial protein and was shown to regulate the mitochondrial membrane potential of prostate cancer cells [25]. We found that MCCC2 restored the mitochondrial membrane potential in cisplatin-induced AKI, suggesting that MCCC2 may inhibit apoptosis by alleviating mitochondrial dysfunction. In addition, mitoROS accounts for a major part of the total ROS in the cell, and cisplatin directly damages mitochondria, resulting in an increase in mitoROS levels and a decrease in ATP levels [43]. However, we found that MCCC2 could reduce intracellular mitoROS levels and increase ATP levels. The effect of MCCC2 on intracellular oxidative stress may arise from the inhibition of mitoROS production. Moreover, MCCC2 can improve the mitochondrial energy supply.

Mitochondria are organelles in a highly dynamic state of flux, and the number of mitochondria is a key factor in maintaining normal mitochondrial function and a key mechanism by which the cell is able to adapt to higher energy demands, while mitochondrial biogenesis is an important determinant of the number of mitochondria [44]. Mitochondrial biogenesis refers to the production of new mitochondria, which are essential for replacing damaged mitochondrial proteins, increasing energy production and decreasing mitoROS levels [45,46]. PGC-1α is a major regulator of mitochondrial biogenesis that is abundantly expressed in the proximal tubules of the kidney [47] and activates a series of transcription factors that promote mtDNA replication and mitochondrial protein expression [48]. In a mouse model of cisplatin-induced AKI, the expression of PGC-1α in renal tissues was found to be significantly decreased [47]. In addition, aldehyde dehydrogenase 2 can alleviate cisplatin- and maleic acid-induced AKI by promoting PGC-1α–mediated mitochondrial biogenesis [19]. Previous studies have also demonstrated that promoting mitochondrial biogenesis could alleviate mitochondrial dysfunction to mitigate cisplatin-induced AKI [20,21]. In our study, we demonstrated that cisplatin decreased the expression of PGC-1α, but this decrease was alleviated by MCCC2. In addition, the expression of mitochondrial genes downstream of PGC-1α decreases in AKI or CKD [20,4951], and we found that MCCC2 increased the expression of these genes. In cisplatin-treated cells, mtDNA copy numbers also decreased [52], which was confirmed in our experiments, while MCCC2 reversed this decrease. In conclusion, these data suggested that mitochondrial biogenesis was suppressed in cisplatin-induced AKI and that MCCC2 alleviated this suppression.

Leucine is an essential amino acid that must be obtained externally, and it is ultimately catabolized to acetoacetate and acetyl coenzyme A in the presence of MCC [53]. In cisplatin-induced AKI, our results showed significant downregulation of MCCC2 expression, suggesting an impairment of leucine catabolism, which is consistent with previous findings [29] and is also supported by a decrease in the acetoacetate content in cisplatin-treated HK-2 cells. Therefore, to explore whether leucine accumulation leads to cellular damage, we treated HK-2 cells with different concentrations of leucine. Surprisingly, even high concentrations of leucine did not damage HK-2 cells, suggesting that a lack of leucine may exacerbate cellular damage. Therefore, we cultured HK-2 cells in a leucine-deprived medium, and the results indicated that leucine deprivation decreased the ability of MCCC2 to alleviate mitochondrial dysfunction and promote mitochondrial biogenesis. We hypothesized that these effects might be related to the products of leucine catabolism, which is supported by previous studies showing that metabolites of leucine (e.g., β-hydroxy-β-methylbutyrate and acetoacetate) upregulate the expression of PGC-1α, promote mitochondrial biogenesis and alleviate mitochondrial dysfunction [5457].

Our study has several limitations. First, MCC deficiency, which leads to abnormal leucine metabolism, causes autosomal recessive disease, therefore, we did not knock down MCCC2 in vivo. Second, although we found that MCCC2 expression was significantly reduced in cisplatin-induced AKI models, we did not explore the reasons for this reduction. Third, our study revealed that leucine catabolism was inhibited in cisplatin-induced AKI, whereas the promotion of leucine catabolism alleviated AKI, suggesting that leucine metabolites may play an important role in this process, which will be a focus of our subsequent studies. Fourth, we found that overexpression of MCCC2 promoted mitochondrial biogenesis and that leucine deprivation reversed this effect, but we did not further explore the reasons for the promotion of mitochondrial biogenesis, which is something that we will continue to explore in the future.

In summary, our study revealed the protective effect of MCCC2 against cisplatin-induced AKI (Fig. 9). Specifically, MCCC2 maintained leucine catabolism, which was inhibited in cisplatin-induced AKI, to promote mitochondrial biogenesis, which can upregulate the expression of mitochondrial genes and mtDNA replication, increasing ATP production, improving the mitochondrial membrane potential, reducing the production of mitoROS, mitigating the destruction of mitochondrial structure, and consequently, alleviating apoptosis and oxidative stress. These findings provide a theoretical direction for the targeted treatment of cisplatin-induced AKI.

Figure 9.

Schematic illustration of the mechanism by which MCCC2 protects kidneys against cisplatin-induced acute kidney injury (AKI).

ATP, adenosine triphosphate; mitoROS, mitochondrial reactive oxygen species; MCCC2, methylcrotonyl‑CoA carboxylase 2; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha.

Notes

Conflicts of interest

All authors have no conflicts of interest to declare.

Funding

This research was funded by the Nature Science Foundation of Guangdong Province, China (project No: 2019A1515012116).

Data sharing statement

The data presented in this study are available from the corresponding author upon reasonable request.

Authors’ contributions

Conceptualization, Methodology: HL, KW, Hao Qi, YD

Data curation: LS, JQ

Formal analysis: HL, KW, Huiyue Qi

Funding acquisition, Project administration, Supervision: YD

Investigation: KW, Huiyue Qi

Validation: HL, KW, Hao Qi, Huiyue Qi, LS, JQ

Visualization: HL, KW

Writing–original draft: HL, Hao Qi

Writing–review & editing: HL, YD

All authors read and approved the final manuscript.

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Figure 1.

MCCC2 was downregulated in cisplatin-induced acute kidney injury.

In vitro, HK-2 cells were treated with different concentrations (0, 10, 20, and 40 μM) of cisplatin. In vivo, mice were injected intraperitoneally with cisplatin (25 mg/kg). (A) Results of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes (DEGs) in the GSE142173 dataset. (B) Results of KEGG enrichment analysis of mitochondrial genes in the MitoCarta 3.0 database (https://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways). (C) Intersection of genes involved in branched-chain amino acids degradation between the GSE142173 DEGs and mitochondrial genes in the MitoCarta 3.0 database. (D) Volcano plot of genes in the intersection group. (E, F) Results of western blot analysis of MCCC2 expression in HK-2 cells (n = 3). (G, H) Results of immunofluorescence staining of MCCC2 in kidney tissue sections from mice (n = 6). Scale bars, 100 μm. (I, J) Results of western blot analysis of MCCC2 expression in kidney tissues of mice (n = 6). (K) To determine the intracellular location of MCCC2, immunofluorescence colocalization of MitoTracker (Beyotime Biotechnology) and MCCC2 was performed. Scale bars, 10 μm. (L, O) Results of real-time quantitative polymerase chain reaction analysis of the efficiency of MCCC2 overexpression and knockdown in HK-2 cells (n = 3). (M, N, P, Q) Results of western blot analysis of the efficiency of MCCC2 overexpression and knockdown in HK-2 cells (n = 3). All the data are presented as the mean ± standard deviation. **p < 0.01, ***p < 0.001.

cis, cisplatin; DAPI, 4′,6-diamidino-2-phenylindole; MCCC2, methylcrotonyl‑CoA carboxylase 2; NS, not significant.

Figure 2.

MCCC2 alleviated cell damage in cisplatin-treated HK-2 cells.

HK-2 cells were treated with 40-μM cisplatin for 24 hours. (A, B) Results of the CCK-8 cell viability assay of MCCC2-overexpressing and MCCC2-knockdown HK-2 cells (n = 5). (C–F) Results of western blot analysis of cleaved caspase-3 levels in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells (n = 3). (G–J) Results of flow cytometry analysis of apoptosis levels in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells (n = 3). (K–N) Results of fluorescence detection of ROS levels in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells by using CM-H2DCFDA (n = 3). Scale bars, 100 μm. The data are presented as the mean ± standard deviation. **p < 0.01, ***p < 0.001.

cis, cisplatin; FITC, fluorescein isothiocyanate; MCCC2, methylcrotonyl‑CoA carboxylase 2; PI, propidium iodide; sh, knockdown; oe, overexpression; NC, control.

Figure 3.

MCCC2 alleviated mitochondrial dysfunction in cisplatin-treated HK-2 cells.

(A–D) Representative images of mitochondrial membrane potential changes in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells, as determined by using a JC-1 kit and quantitative analysis. Scale bars, 50 μm. (E, F) Adenosine triphosphate (ATP) levels in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells. (G–J) Representative images and the results of quantitative analysis of MitoSOX (Dojindo Laboratories) staining of MCCC2-overexpressing and MCCC2-knockdown HK-2 cells. Scale bars, 50 μm. All the data are presented as the mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

cis, cisplatin; JC-1, J-aggregate-1; MCCC2, methylcrotonyl‑CoA carboxylase 2; sh, knockdown; oe, overexpression; NC, control.

Figure 4.

MCCC2 promoted mitochondrial biogenesis in cisplatin-treated HK-2 cells.

(A–D) Results of western blot analysis of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells. (E–L) Results of real-time quantitative polymerase chain reaction analysis of the messenger RNA (mRNA) levels of ATP5a1, NDUFS1, NDUFB8, and SDHB in MCCC2-overexpressing (E–H) and MCCC2-knockdown (I–L) HK-2 cells. (M–P) Representative immunofluorescence images and the results of quantitative analysis of translocase of outer mitochondrial membrane 20 (TOMM20) expression in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells. Scale bars, 50 μm. (Q, R) Mitochondrial DNA (mtDNA) copy numbers in MCCC2-overexpressing and MCCC2-knockdown HK-2 cells. All the data are presented as the mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

cis, cisplatin; MCCC2, methylcrotonyl‑CoA carboxylase 2; sh, knockdown; oe, overexpression; NC, control.

Figure 5.

Leucine deprivation reversed the effects of MCCC2 overexpression on mitochondrial function and biogenesis.

(A) Results of the measurement of the intracellular acetoacetate content (n = 3). (B) HK-2 cells were treated with different concentrations (0, 0.25, 0.5, 1, 2, 3, 4, 6, and 8 μM) of leucine for 24 hours, after which cell viability was assayed (n = 5). (C) Cell viability after leucine deprivation (n = 5). (D) Intracellular adenosine triphosphate (ATP) content after leucine deprivation (n = 3). (E, F) Results of western blot analysis of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression after leucine deprivation (n = 3). (G) Mitochondrial DNA (mtDNA) copy numbers after leucine deprivation (n = 3). (H, I) Results of immunofluorescence staining of translocase of outer mitochondrial membrane 20 (TOMM20) after leucine deprivation (n = 3). Scale bars, 50 μm. All the data are presented as the mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.

cis, cisplatin; DAPI, 4′,6-diamidino-2-phenylindole; leu, leucine; MCCC2, methylcrotonyl‑CoA carboxylase 2; NS, not significant; oe, overexpression; NC, control.

Figure 6.

Overexpression of MCCC2 alleviated renal dysfunction and tissue injury in mice with cisplatin-induced acute kidney injury (AKI).

(A) Schematic diagram of the establishment of the cisplatin-induced AKI mouse model. (B, C) Results of western blot analysis of the efficiency of MCCC2 overexpression in mouse kidneys. Renal function was assessed by the levels of (D) serum creatinine and (E) urea nitrogen. (F, G) Results of real-time quantitative polymerase chain reaction analysis of the messenger RNA (mRNA) levels of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). (H, I) Representative images of hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining to assess kidney tissue damage (Scale bars, 20 μm). (J) Quantitative data of tubular injury. The data are presented as the mean ± standard deviation (n = 6). **p < 0.01, ***p < 0.001.

AAV, adeno-associated virus; cis, cisplatin; ip, intraperitoneal injection; MCCC2, methylcrotonyl‑CoA carboxylase 2; NC, control.

Figure 7.

Overexpression of MCCC2 alleviated cell apoptosis and oxidative stress in mice with cisplatin-induced acute kidney injury.

(A, B) Results of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining to detect apoptosis in renal tissues (green represents positive cells). Scale bars, 100 μm. (C, D) Results of western blot analysis of cleaved caspase-3 levels in renal tissues. (E) Malondialdehyde (MDA) levels in renal tissues. (F, G) Results of immunofluorescence staining for 4-HNE in kidney sections. Scale bars, 100 μm. All the data are presented as the mean ± standard deviation (n = 6). ***p < 0.001.

AAV, adeno-associated virus; cis, cisplatin; DAPI, 4′,6-diamidino-2-phenylindole; HPF, high-power field; MCCC2, methylcrotonyl‑CoA carboxylase 2; MDA, malondialdehyde; 4-HNE, 4-hydroxynonenal; NC, control.

Figure 8.

Effects of MCCC2 overexpression on mitochondrial function and biogenesis in mice with cisplatin-induced acute kidney injury.

(A) Representative transmission electron microscope (TEM) images of the mitochondrial structure in renal tubular epithelial cells. Scale bars, 1 μm. (B, C) Results of MitoSOX (Dojindo Laboratories) staining of kidney sections (n = 6). Scale bars, 100 μm. (D) Adenosine triphosphate (ATP) levels measured in renal tissues (n = 6). (E, F) Results of western blot analysis of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression in kidney tissues (n = 6). (G–J) Real-time quantitative polymerase chain reaction analysis was performed to detect the messenger RNA (mRNA) levels of ATP5a1, NDUFS1, NDUFB8, and SDHB in renal tissues (n = 6). (K, L) Results of translocase of outer mitochondrial membrane 20 (TOMM20) staining in kidney sections (n = 6). Scale bars, 100 μm. (M) Mitochondrial DNA (mtDNA) copy numbers in kidney tissues (n = 6). All the data are presented as the mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.

AAV, adeno-associated virus; cis, cisplatin; DAPI, 4′,6-diamidino-2-phenylindole; MCCC2, methylcrotonyl‑CoA carboxylase 2; NC, control.

Figure 9.

Schematic illustration of the mechanism by which MCCC2 protects kidneys against cisplatin-induced acute kidney injury (AKI).

ATP, adenosine triphosphate; mitoROS, mitochondrial reactive oxygen species; MCCC2, methylcrotonyl‑CoA carboxylase 2; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha.